ON Retinal Ganglion Cells Research Articles

Electrically-evoked responses for retinal prostheses are differentially altered depending on ganglion cell types in outer retinal neurodegeneration caused by Crb1 gene mutation.

Microelectronic prostheses for artificial vision stimulate neurons surviving outer retinal neurodegeneration such as retinitis pigmentosa (RP). Yet, the quality of prosthetic vision substantially varies across subjects, maybe due to different levels of retinal degeneration and/or distinct genotypes. Although the RP genotypes are remarkably diverse, prosthetic studies have primarily used retinal degeneration (rd) 1 and 10 mice, which both have Pde6b gene mutation. Here, we report the electric responses arising in retinal ganglion cells (RGCs) of the rd8 mouse model which has Crb1 mutation. We first investigated age-dependent histological changes of wild-type (wt), rd8, and rd10 mice retinas by H&E staining. Then, we used cell-attached patch clamping to record spiking responses of ON, OFF and direction selective (DS) types of RGCs to a 4-ms-long electric pulse. The electric responses of rd8 RGCs were analyzed in comparison with those of wt RGCs in terms of individual RGC spiking patterns, populational characteristics, and spiking consistency across trials. In the histological examination, the rd8 mice showed partial retinal foldings, but the outer nuclear layer thicknesses remained comparable to those of the wt mice, indicating the early-stage of RP. Although spiking patterns of each RGC type seemed similar to those of the wt retinas, correlation levels between electric vs. light response features were different across the two mouse models. For example, in comparisons between light vs. electric response magnitudes, ON/OFF RGCs of the rd8 mice showed the same/opposite correlation polarity with those of wt mice, respectively. Also, the electric response spike counts of DS RGCs in the rd8 retinas showed a positive correlation with their direction selectivity indices (r = 0.40), while those of the wt retinas were negatively correlated (r = -0.90). Lastly, the spiking timing consistencies of late responses were largely decreased in both ON and OFF RGCs in the rd8 than the wt retinas, whereas no significant difference was found across DS RGCs of the two models. Our results indicate the electric response features are altered depending on RGC types even from the early-stage RP caused by Crb1 mutation. Given the various degeneration patterns depending on mutation genes, our study suggests the importance of both genotype- and RGC type-dependent analyses for retinal prosthetic research.

Short pulses of epiretinal prostheses evoke network-mediated responses in retinal ganglion cells by stimulating presynaptic neurons

Objective. Microelectronic retinal implant aims to restore functional vision with electric stimulation. Short pulses are generally known to directly activate retinal ganglion cells (RGCs) with a notion of one or two spike(s) per pulse. In the present work, we systematically explore network-mediated responses that arise from various short pulses in both normal and degenerate retinas. Approach. Cell-attached patch clamping was used to record spiking responses of RGCs in wild-type (C57BL/6J) and retinal degeneration (rd10) mice. Alpha RGCs of the mouse retinas were targeted by their large soma sizes and classified by their responses to spot flashes. Then, RGCs were electrically stimulated by various conditions such as duration (100–460 μs), count (1–10), amplitude (100–400 μA), and repeating frequency (10–40 Hz) of short pulses. Also, their responses were compared with each own response to a single 4 ms long pulse which is known to evoke strong indirect responses. Main results. Short pulses evoked strong network-mediated responses not only in both ON and OFF types of RGCs in the healthy retinas but also in RGCs of the severely degenerate retina. However, the spike timing consistency across repeats not decreased significantly in the rd10 RGCs compared to the healthy ON and OFF RGCs. Network-mediated responses of ON RGCs were highly dependent on the current amplitude of stimuli but much less on the pulse count and the repetition frequency. In contrast, responses of OFF RGCs were more influenced by the number of stimuli than the current amplitude. Significance. Our results demonstrate that short pulses also elicit indirect responses by activating presynaptic neurons. In the case of the commercial retinal prostheses using repeating short pulses, there is a possibility that the performance of clinical devices is highly related to the preserved retinal circuits. Therefore, examination of surviving retinal neurons in patients would be necessary to improve the efficacy of retinal prostheses.

Electric Stimulation Elicits Heterogeneous Responses in ON but Not OFF Retinal Ganglion Cells to Transmit Rich Neural Information.

Retinal implants electrically stimulate surviving retinal neurons to restore vision in people blinded by outer retinal degeneration. Although the healthy retina is known to transmit a vast amount of visual information to the brain, it has not been studied whether prosthetic vision contains a similar amount of information. Here, we assessed the neural information transmitted by population responses arising in brisk transient (BT) and brisk sustained (BS) subtypes of ON and OFF retinal ganglion cells (RGCs) in the rabbit retina. To correlate the response heterogeneity and the information transmission, we first quantified the cell-to-cell heterogeneity by calculating the spike time tiling coefficient (STTC) across spiking patterns of RGCs in each type. Then, we computed the neural information encoded by the RGC population in a given type. In responses to light stimulation, spiking activities were more heterogeneous in OFF than ON RGCs (STTCAVG = 0.36, 0.45, 0.77 and 0.55 for OFF BT, OFF BS, ON BT, and ON BS, respectively). Interestingly, however, in responses to electric stimulation, both BT and BS subtypes of OFF RGCs showed remarkably homogeneous spiking patterns across cells (STTCAVG = 0.93 and 0.82 for BT and BS, respectively), whereas the two subtypes of ON RGCs showed slightly increased populational heterogeneity compared to light-evoked responses (STTCAVG = 0.71 and 0.63 for BT and BS, respectively). Consequently, the neural information encoded by the electrically-evoked responses of a population of 15 RGCs was substantially lower in the OFF than the ON pathway: OFF BT and BS cells transmit only ~23% and ~53% of the neural information transmitted by their ON counterparts. Together with previously-reported natural spiking activities in ON RGCs, the higher neural information may make ON responses more recognizable, eliciting the biased percepts of bright phosphenes.

Probing ON and OFF Retinal Pathways in Glaucoma Using Electroretinography.

Glaucoma is a progressive neurodegenerative disease involving damage and eventually death of retinal ganglion cells (RGCs) that comprise the optic nerve. This review summarizes current understanding of specific RGC type vulnerability in glaucoma and how electroretinography (ERG) may provide an objective measure of these functional perturbations. There is building evidence to suggest that ON RGCs, which respond to light increments, may be more resilient to elevated intraocular pressure and glaucoma, whereas OFF RGCs, which respond to light decrements, may be more susceptible. ERG experiments in nonhuman primates and mice have also shown that the ON- and OFF-pathways can be separated using a variety of techniques such as pattern ERG and the photopic negative response. Another ERG paradigm of interest to separate the ON and OFF responses is a flicker stimulus at varying temporal frequencies. Response to lower temporal frequencies is associated with the ON-pathway, and ERG response to higher frequencies is associated with the OFF-pathway. In mice, experimental glaucoma models have shown greater decreases in ERG response at higher frequencies, suggesting that the OFF-pathway is more susceptible. We also summarize current clinical ERG protocols used for glaucoma and discuss innovations for developing new types of stimuli that can further separate the ON- and OFF-pathways. Applying these novel paradigms that distinguish ON- and OFF-pathways may ultimately improve glaucoma diagnostics and monitoring of glaucoma progression.Translational RelevanceBased on our current understanding of specific RGC type vulnerability in glaucoma, we explore how ERG may provide an objective measure of ON- versus OFF-pathway functional perturbations.

Functional-pathway-dominant contrast adaptation and sensitization in mouse retinal ganglion cells.

Retinal ganglion cells (RGCs) reduce their light sensitivity during persistent high-contrast stimulation to prevent saturation to strong inputs and improve coding efficiency. This process is known as contrast adaptation. However, contrast adaptation also reduces RGCs' light response to weak inputs. On the other hand, some RGCs undergo contrast sensitization, and these RGCs respond to weak inputs following high contrast stimulation. In the present study, multi-electrode recordings were conducted on isolated mouse retinas under full-field visual stimulation with different contrast levels. Adaptation and sensitization were mainly observed in OFF and ON pathways, respectively. The results of linear-nonlinear analysis and stimulus reconstruction revealed that both the light sensitivity and encoded information were changed in opposite directions in adaptation and sensitization processes. Our work suggests that contrast adaptation and sensitization are two opposite dynamic processes. In mouse retina, OFF RGCs utilize adaptation to increase the discrimination of strong OFF inputs. On the other hand, ON RGCs use sensitization to increase the sensitivity to weak ON inputs. This functional differentiation might be meaningful for the mouse's survival as it lives in environments in which strong OFF stimuli often indicate potential predators while weak ON stimuli are usually related to movement and might be important for predation.

Scaling of the AIS and Somatodendritic Compartments in α S RGCs.

The anatomical properties of the axon initial segment (AIS) are tailored in certain types of CNS neurons to help optimize different aspects of neuronal function. Here, we questioned whether the AISs of retinal ganglion cells (RGC) were similarly customized, and if so, whether they supported specific RGC functions. To explore this, we measured the AIS properties in alpha sustained RGCs (α S RGCs) of mouse; α S RGCs sizes vary systematically along the nasal temporal axis of the retina, making these cells an attractive population with which to study potential correlations between AIS properties and cell size. Measurements of AIS length as well as distance from the soma revealed that both were scaled to cell size, i.e., cells with large dendritic fields had long AISs that were relatively far from the soma. Within the AIS, the percentage of Nav1.6 voltage-gated sodium channels remained highly consistent, regardless of cell size or other AIS properties. Although ON RGCs were slightly larger than OFF cells at any given location of the retina, the level of scaling and relative distribution of voltage-gated sodium channels were highly similar. Computational modeling revealed that AIS scaling influenced spiking thresholds, spike rate as well as the kinetics of individual action potentials, Interestingly, the effect of individual features of the AIS varied for different neuronal functions, e.g., AIS length had a larger effect on the efficacy by which the AIS initiated spike triggered the somatic spike than it did on repetitive spiking. The polarity of the effect varied for different properties, i.e., increases to soma size increased spike threshold while increases to AIS length decreased threshold. Thus, variations in the relative level of scaling for individual components could fine tune threshold or other neuronal functions. Light responses were highly consistent across the full range of cell sizes suggesting that scaling may post-synaptically shape response stability, e.g., in addition to several well-known pre-synaptic contributors.

Optimal Electric Stimulus Amplitude Improves the Selectivity Between Responses of ON Versus OFF Types of Retinal Ganglion Cells.

Outer retinal degenerative diseases destroy photoreceptors primarily in the retina, resulting in a profound vision loss. Fortunately, surviving retinal neurons can be electrically stimulated using retinal prostheses to re-transmit visual information. Although retinal prostheses are promising for sight restoration, the best performance is still sub-optimal. For the enhanced performance, it is critical to optimize stimulation parameters. This study explored how stimulus charge and current amplitude alters responses of retinal ganglion cells (RGCs). From the isolated mouse retina, spiking activities of alpha ON, OFF sustained, and OFF transient RGCs were recorded in response to epiretinally-delivered cathodal current pulses ranging from -10 to [Formula: see text]. We have found that intermediate current amplitudes (-30 and [Formula: see text]) maximize the response ratio of ON over OFF types. Also, the ON/OFF response ratio was always bigger for 10-ms-long than 5-ms-long stimuli. It was because, by the longer pulses, ON RGC responses were always significantly enhanced ( ) while OFF RGC responses were minimally changed ( ). Given the earlier work reporting electrically-elicited responses are more natural in ON than OFF RGCs, the present study suggests effect strategies of more selective activation of ON RGCs for improved efficacy of retinal implant.

Inhibition of non-NMDA ionotropic glutamate receptors delays the retinal degeneration in rd10 mouse

Retinitis pigmentosa (RP) is a hereditary blinding disease characterized by neurodegeneration of photoreceptors. Retinal ganglion cells (RGCs) in animal models of RP exhibit an abnormally high spontaneous activity that interferes with signal processing. Blocking AMPA/Kainate receptors by bath application of CNQX decreases the spontaneous firing, suggesting that inhibiting these receptors in vivo may help maintain the function of inner retinal neurons in rd10 mice experiencing photoreceptor degeneration. To test this, rd10 mice were i.p. injected with CNQX or GYKI 52466 (an AMPA receptor antagonist) for 1–2 weeks, and examined for their retinal morphology (by immunocytochemistry), function (by MEA recordings) and visual behaviors (using a black/white box). Our data show that iGluRs were up-regulated in the inner plexiform layer (IPL) of rd10 retinas. Application of CNQX at low doses both in vitro and in vivo, attenuated the abnormal spontaneous spiking in RGCs, and increased the light-evoked response of ON RGCs, whereas GYKI 52466 had little effect. CNQX application also improved the behavioral performance. Interestingly, in vivo administration of CNQX delayed photoreceptor degeneration, evidenced by the increased cell number and restored structure. CNQX also improved the structure of bipolar cells. Together, we demonstrated that during photoreceptor degeneration, blockade of the non-NMDA iGluRs decelerates the progression of RGCs dysfunction, possibly by dual mechanisms including slowing photoreceptor degeneration and modulating signal processing within the IPL. Accordingly, this strategy may effectively extend the time window for treating RP.

Differential electrical responses in retinal ganglion cell subtypes: effects of synaptic blockade and stimulating electrode location

Objective. Visual prostheses have shown promising results in restoring visual perception to blind patients. The ability to differentially activate retinal ganglion cell (RGC) subtypes could further improve the efficacy of these medical devices. Approach. Using whole-cell patch clamp, we investigated membrane potential differences between ON and OFF RGCs in the mouse retina when their synaptic inputs were blocked by synaptic blockers, and examined the differences in stimulation thresholds under such conditions. By injecting intracellular current, we further confirmed the relationship between RGC stimulation thresholds and resting membrane potentials (RMPs). In addition, we investigated the effects of stimulating electrode location on the differences in stimulation thresholds between ON and OFF RGCs. Main results. With synaptic blockade, ON RGCs became significantly more hyperpolarized (from −61.8 ± 1.4 mV to −70.8 ± 1.6 mV), while OFF RGCs depolarized slightly (from −60.5 ± 0.7 mV to −58.6 ± 0.9 mV). RGC stimulation thresholds were negatively correlated with their RMPs (Pearson r value: −0.5154; p-value: 0.0042). Thus, depriving ON RGCs of synaptic inputs significantly increased their thresholds (from 14.7 ± 1.3 µA to 22.3 ± 2.1 µA) over those of OFF RGCs (from 13.2 ± 0.7 µA to 13.1 ± 1.1 µA). However, with control solution, ON and OFF RGC stimulation thresholds were not significantly different. Finally, placement of the stimulating electrode away from the axon enhanced differences in stimulation thresholds between ON and OFF RGCs, facilitating preferential activation of OFF RGCs. Significance. Since ON and OFF RGCs have antagonistic responses to natural light, achieving differential RGC activation could convey more natural visual information, leading to better visual prosthesis outcomes.

Closed-Loop Efficient Searching of Optimal Electrical Stimulation Parameters for Preferential Excitation of Retinal Ganglion Cells.

The ability for visual prostheses to preferentially activate functionally-distinct retinal ganglion cells (RGCs) is important for improving visual perception. This study investigates the use of high frequency stimulation (HFS) to elicit RGC activation, using a closed-loop algorithm to search for optimal stimulation parameters for preferential ON and OFF RGC activation, resembling natural physiological neural encoding in response to visual stimuli. We evaluated the performance of a wide range of electrical stimulation amplitudes and frequencies on RGC responses in vitro using murine retinal preparations. It was possible to preferentially excite either ON or OFF RGCs by adjusting amplitudes and frequencies in HFS. ON RGCs can be preferentially activated at relatively higher stimulation amplitudes (>150 μA) and frequencies (2–6.25 kHz) while OFF RGCs are activated by lower stimulation amplitudes (40–90 μA) across all tested frequencies (1–6.25 kHz). These stimuli also showed great promise in eliciting RGC responses that parallel natural RGC encoding: ON RGCs exhibited an increase in spiking activity during electrical stimulation while OFF RGCs exhibited decreased spiking activity, given the same stimulation amplitude. In conjunction with the in vitro studies, in silico simulations indicated that optimal HFS parameters could be rapidly identified in practice, whilst sampling spiking activity of relevant neuronal subtypes. This closed-loop approach represents a step forward in modulating stimulation parameters to achieve appropriate neural encoding in retinal prostheses, advancing control over RGC subtypes activated by electrical stimulation.

Electric stimulus duration alters network-mediated responses depending on retinal ganglion cell type

Objective. To improve the quality of artificial vision that arises from retinal prostheses, it is important to bring electrically-elicited neural activity more in line with the physiological signaling patterns that arise normally in the healthy retina. Our previous study reported that indirect activation produces a closer match to physiological responses in ON retinal ganglion cells (RGCs) than in OFF cells (Im and Fried 2015 J. Physiol. 593 3677–96). This suggests that a preferential activation of ON RGCs would shape the overall retinal response closer to natural signaling. Recently, we found that changes to the rate at which stimulation was delivered could bias responses towards a stronger ON component (Im and Fried 2016a J. Neural Eng. 13 025002), raising the possibility that changes to other stimulus parameters can similarly bias towards stronger ON responses. Here, we explore the effects of changing stimulus duration on the responses in ON and OFF types of brisk transient (BT) and brisk sustained (BS) RGCs. Approach. We used cell-attached patch clamp to record RGC spiking in the isolated rabbit retina. Targeted RGCs were first classified as ON or OFF type by their light responses, and further sub-classified as BT or BS types by their responses to both light and electric stimuli. Spiking in targeted RGCs was recorded in response to electric pulses with durations varying from 5 to100 ms. Stimulus amplitude was adjusted at each duration to hold total charge constant for all experiments. Main results. We found that varying stimulus durations modulated responses differentially for ON versus OFF cells: in ON cells, spike counts decreased significantly with increasing stimulus duration while in OFF cells the changes were more modest. The maximum ratio of ON versus OFF responses occurred at a duration of ~10 ms. The difference in response strength for BT versus BS cells was much larger in ON cells than in OFF cells. Significance. The stimulation rates preferred by subjects during clinical trials are similar to the rates that maximize the ON/OFF response ratio in in vitro testing (Im and Fried 2016a J. Neural Eng. 13 025002). Here, we determine the stimulus duration that produces the strongest bias towards ON responses and speculate that it will further enhance clinical effectiveness.

High glucose levels impact visual response properties of retinal ganglion cells in C57 mice-An in vitro physiological study.

This study investigated visual response properties of retinal ganglion cells (RGCs) under high glucose levels. Extracellular single-unit responses of RGCs from mouse retinas were recorded. And the eyecup was prepared as a flat mount in a recording chamber and superfused with Ames medium. The averaged RF size of the ON RGCs (34.1±2.9, n=14) was significantly smaller than the OFF RGCs under the HG (49.3±0.3, n=12) (P<0.0001) conditions. The same reduction pattern was also observed in the osmotic control group (HM) between ON and OFF RGCs (P<0.0001). The averaged luminance threshold (LT) of ON RGCs increased significantly under HG or HM (HG: P<0.0001; HM: P<0.0002). OFF RGCs exhibited a similar response pattern under the same conditions (HG: P<0.01; HM: P<0.0002). The averaged contrast gain of ON cells was significantly lower than that of OFF cells with the HM treatment (P<0.015, unpaired Student's t test). The averaged contrast gain of ON cells was significantly higher than OFF cells with the HG treatment (P<0.0001). The present results suggest that HG reduced receptive field center size, suppressed luminance threshold, and attenuated contrast gain of RGCs. The impact of HG on ON and OFF RGCs may be mediated via different mechanisms.

Spatiotemporal characteristics of retinal response to network-mediated photovoltaic stimulation.

Subretinal prostheses aim at restoring sight to patients blinded by photoreceptor degeneration using electrical activation of the surviving inner retinal neurons. Today, such implants deliver visual information with low-frequency stimulation, resulting in discontinuous visual percepts. We measured retinal responses to complex visual stimuli delivered at video rate via a photovoltaic subretinal implant and by visible light. Using a multielectrode array to record from retinal ganglion cells (RGCs) in the healthy and degenerated rat retina ex vivo, we estimated their spatiotemporal properties from the spike-triggered average responses to photovoltaic binary white noise stimulus with 70-μm pixel size at 20-Hz frame rate. The average photovoltaic receptive field size was 194 ± 3 μm (mean ± SE), similar to that of visual responses (221 ± 4 μm), but response latency was significantly shorter with photovoltaic stimulation. Both visual and photovoltaic receptive fields had an opposing center-surround structure. In the healthy retina, ON RGCs had photovoltaic OFF responses, and vice versa. This reversal is consistent with depolarization of photoreceptors by electrical pulses, as opposed to their hyperpolarization under increasing light, although alternative mechanisms cannot be excluded. In degenerate retina, both ON and OFF photovoltaic responses were observed, but in the absence of visual responses, it is not clear what functional RGC types they correspond to. Degenerate retina maintained the antagonistic center-surround organization of receptive fields. These fast and spatially localized network-mediated ON and OFF responses to subretinal stimulation via photovoltaic pixels with local return electrodes raise confidence in the possibility of providing more functional prosthetic vision. NEW & NOTEWORTHY Retinal prostheses currently in clinical use have struggled to deliver visual information at naturalistic frequencies, resulting in discontinuous percepts. We demonstrate modulation of the retinal ganglion cells (RGC) activity using complex spatiotemporal stimuli delivered via subretinal photovoltaic implant at 20 Hz in healthy and in degenerate retina. RGCs exhibit fast and localized ON and OFF network-mediated responses, with antagonistic center-surround organization of their receptive fields.

Elevated IOP alters the space–time profiles in the center and surround of both ON and OFF RGCs in mouse

Glaucoma is a leading cause of blindness worldwide, and is characterized by progressive retinal ganglion cell (RGC) death. An experimental model of glaucoma has been established by elevating the intraocular pressure (IOP) via microbead occlusion of ocular fluid outflow in mice. Studies in this model have found visual dysfunction that varied with adaptational state, occurred before anatomical changes, and affected OFF RGCs more than ON RGCs. These results indicate subtle alterations in the underlying retinal circuitry that could help identify disease before irreversible RGC changes. Therefore, we looked at how RGC function was altered with elevated IOP under both photopic and scotopic conditions. We first found that responses to light offset are diminished with IOP elevation along with a concomitant decrease in receptive field center size for OFF RGCs. In addition, the antagonistic surround strength and size was reduced in ON RGCs. Furthermore, elevation of IOP significantly accelerated the photopic temporal tuning of RGC center responses in both ON and OFF RGCs. We found that some of the IOP-induced functional changes to OFF RGCs relied on ON cross-over pathways, indicating dysfunction in inner retinal circuitry. Overall, these results suggest that IOP alters multiple functions in the retina depending on the adaptational state. They provide a basis for designing multiple functional tests for early detection of glaucoma and for circuit-specific therapeutic targets in treatment of this blinding disease.

Loss of Neuroligin3 specifically downregulates retinal GABAAα2 receptors without abolishing direction selectivity.

The postsynaptic adhesion proteins Neuroligins (NLs) are essential for proper synapse function, and their alterations are associated with a variety of neurodevelopmental disorders. It is increasingly clear that each NL isoform occupies specific subsets of synapses and is able to regulate the function of discrete networks. Studies of NL2 and NL4 in the retina in particular have contributed towards uncovering their role in inhibitory synapse function. In this study we show that NL3 is also predominantly expressed at inhibitory postsynapses in the retinal inner plexiform layer (IPL), where it colocalizes with both GABAA- and glycinergic receptor clusters in a 3:2 ratio. In the NL3 deletion-mutant (knockout or KO) mouse, we uncovered a dramatic reduction of the number of GABAAα2-subunit containing GABAA receptor clusters at the IPL. Retinal activity was thereafter assessed in KO and wild-type (WT) littermates by multi-electrode-array recordings of the output cells of retina, the retinal ganglion cells (RGCs). RGCs in the NL3 KO showed reduced spontaneous activity and an altered response to white noise stimulation. Moreover, upon application of light flashes, the proportion of cells firing at light offset (OFF RGCs) was significantly lower in the NL3 KO compared to WT littermates, whereas the relative number of cells firing at light onset (ON RGCs) increased. Interestingly, although GABAAα2-bearing receptors have been related to direction-selective circuits of the retina, features of direction selective-retinal ganglion cells recorded remained unperturbed in the NL3 KO. Together our data underscore the importance of NL3 for the integrity of specific GABAAergic retinal circuits and identifies NL3 as an important regulator of retinal activity.

Differential stimulation of the retina with subretinally injected exogenous neurotransmitter: A biomimetic alternative to electrical stimulation.

Subretinal stimulation of the retina with neurotransmitters, the normal means of conveying visual information, is a potentially better alternative to electrical stimulation widely used in current retinal prostheses for treating blindness from photoreceptor degenerative diseases. Yet, no subretinal electrical or chemical stimulation study has stimulated the OFF and ON pathways differentially through inner retinal activation. Here, we demonstrate the feasibility of differentially stimulating retinal ganglion cells (RGCs) through the inner nuclear layer of the retina with glutamate, a primary neurotransmitter chemical, in a biomimetic way. We show that controlled pulsatile delivery of glutamate into the subsurface of explanted wild-type rat retinas elicits highly localized simultaneous inhibitory and excitatory spike rate responses in OFF and ON RGCs. We also present the spatiotemporal characteristics of RGC responses to subretinally injected glutamate and the therapeutic stimulation parameters. Our findings could pave the way for future development of a neurotransmitter-based subretinal prosthesis offering more naturalistic vision and better visual acuity than electrical prostheses.

ON and OFF retinal ganglion cells differentially regulate serotonergic and GABAergic activity in the dorsal raphe nucleus.

The dorsal raphe nucleus (DRN), the major source of serotonergic input to the forebrain, receives excitatory input from the retina that can modulate serotonin levels and depressive-like behavior. In the Mongolian gerbil, retinal ganglion cells (RGCs) with alpha-like morphological and Y-like physiological properties innervate the DRN with ON DRN-projecting RGCs out numbering OFF DRN-projecting RGCs. The DRN neurons targeted by ON and OFF RGCs are unknown. To explore retino-raphe anatomical organization, retinal afferents labeled with Cholera toxin B were examined for association with the postsynaptic protein PSD-95. Synaptic associations between retinal afferents and DRN serotonergic and GABAergic neurons were observed. To explore retino-raphe functional organization, light-evoked c-fos expression was examined. Light significantly increased the number of DRN serotonergic and GABAergic cells expressing c-Fos. When ON RGCs were rendered silent while enhancing the firing rate of OFF RGCs, c-Fos expression was greatly increased in DRN serotonergic neurons suggesting that OFF DRN-projecting RGCs predominately activate serotonergic neurons whereas ON DRN-projecting RGCs mainly target GABAergic neurons. Direct glutamatergic retinal input to DRN 5-HT neurons contributes to the complex excitatory drive regulating these cells. Light, via the retinoraphe pathway can modify DRN 5-HT neuron activity which may play a role in modulating affective behavior.

Changes in ganglion cells during retinal degeneration

Inherited retinal degeneration such as retinitis pigmentosa (RP) is associated with photoreceptor loss and concomitant morphological and functional changes in the inner retina. It is not known whether these changes are associated with changes in the density and distribution of synaptic inputs to retinal ganglion cells (RGCs). We quantified changes in ganglion cell density in rd1 and age-matched C57BL/6J-(wildtype, WT) mice using the immunocytochemical marker, RBPMS. Our data revealed that following complete loss of photoreceptors, (∼3months of age), there was a reduction in ganglion cell density in the peripheral retina. We next examined changes in synaptic inputs to A type ganglion cells by performing double labeling experiments in mice with the ganglion cell reporter lines, rd1-Thy1 and age-matched wildtype-Thy1. Ribbon synapses were identified by co-labelling with CtBP2 (RIBEYE) and conventional synapses with the clustering molecule, gephyrin. ON RGCs showed a significant reduction in RIBEYE-immunoreactive synapse density while OFF RGCs showed a significant reduction in the gephyrin-immmunoreactive synapse density. Distribution patterns of both synaptic markers across the dendritic trees of RGCs were unchanged. The change in synaptic inputs to RGCs was associated with a reduction in the number of immunolabeled rod bipolar and ON cone bipolar cells. These results suggest that functional changes reported in ganglion cells during retinal degeneration could be attributed to loss of synaptic inputs.

Physiological and morphological characterization of ganglion cells in the salamander retina

Retinal ganglion cells (RGCs) integrate visual information from the retina and transmit collective signals to the brain. A systematic investigation of functional and morphological characteristics of various types of RGCs is important to comprehensively understand how the visual system encodes and transmits information via various RGC pathways. This study evaluated both physiological and morphological properties of 67 RGCs in dark-adapted flat-mounted salamander retina by examining light-evoked cation and chloride current responses via voltage-clamp recordings and visualizing morphology by Lucifer yellow fluorescence with a confocal microscope. Six groups of RGCs were described: asymmetrical ON–OFF RGCs, symmetrical ON RGCs, OFF RGCs, and narrow-, medium- and wide-field ON–OFF RGCs. Dendritic field diameters of RGCs ranged 102–490μm: narrow field (<200μm, 31% of RGCs), medium field (200–300μm, 45%) and wide field (>300μm, 24%). Dendritic ramification patterns of RGCs agree with the sublamina A/B rule. 34% of RGCs were monostratified, 24% bistratified and 42% diffusely stratified. 70% of ON RGCs and OFF RGCs were monostratified. Wide-field RGCs were diffusely stratified. 82% of RGCs generated light-evoked ON–OFF responses, while 11% generated ON responses and 7% OFF responses. Response sensitivity analysis suggested that some RGCs obtained separated rod/cone bipolar cell inputs whereas others obtained mixed bipolar cell inputs. 25% of neurons in the RGC layer were displaced amacrine cells. Although more types may be defined by more refined classification criteria, this report is to incorporate more physiological properties into RGC classification.

Spontaneous oscillatory activity in rd1 mouse retina is transferred from ON pathway to OFF pathway via glycinergic synapse.

Retinal ganglion cells (RGCs) spike randomly in the dark and carry information about visual stimuli to the brain via specific spike patterns. However, following photoreceptor loss, both ON and OFF type of RGCs exhibit spontaneous oscillatory spike activity, which reduces the quality of information they can carry. Furthermore, it is not clear how the oscillatory activity would interact with the experimental treatment approaches designed to produce artificial vision. The oscillatory activity is considered to originate in ON-cone bipolar cells, AII amacrine cells, and/or their synaptic interactions. However, it is unknown how the oscillatory activity is generated in OFF RGCs. We tested the hypothesis that oscillatory activity is transferred from the ON pathway to the OFF pathway via the glycinergic AII amacrine cells. Using extracellular loose-patch and whole cell patch recordings, we recorded oscillatory activity in ON and OFF RGCs and studied their response to strychnine, a specific glycine receptor blocker. The cells were labeled with a fluorescent dye, and their dendritic stratification in inner plexiform layer was studied using confocal microscopy. Application of strychnine resulted in abolition of the oscillatory burst activity in OFF RGCs but not in ON RGCs, implying that oscillatory activity is generated in ON pathway and is transferred to OFF pathway, likely via the glycinergic AII amacrine cells. We found oscillatory activity in RGCs as early as postnatal day 12 in rd1 mouse, when rod degeneration has started but cones are still intact. This suggests that the oscillatory activity in rd1 mouse retina originates in rod pathway.